Compositions and methods for increasing muscle mass and muscle strength by specifically antagonizing gdf8 and or activin a

ABSTRACT

The present invention provides compositions and methods which involve specifically antagonizing GDF8 and Activin A. In certain embodiments, compositions are provided which comprise a GDF8-specific binding protein and an Activin A-specific binding protein. For example, the invention includes compositions comprising an anti-GDF8 antibody and an anti-Activin A antibody. In other embodiments, antigen-binding molecules are provided which comprise a GDF8-specific binding domain and an Activin A-specific binding domain. For example, the invention includes bispecific antibodies that bind GDF8 and Activin A. The compositions of the present invention are useful for the treatment of diseases and conditions characterized by reduced muscle mass or strength, as well as other conditions which are treatable by antagonizing GDF8 and/or Activin A activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application No. 61/559,175, filed on Nov. 14, 2011;61/607,024, filed on Mar. 6, 2012; and 61/661,451, filed on Jun. 19,2012, the disclosures of which are herein incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for increasingmuscle mass and muscle strength in a subject. More specifically, theinvention relates to compositions that specifically bind GDF8 andActivin A and the use of such compositions to treat diseases anddisorders characterized by decreased muscle mass or strength.

BACKGROUND

Growth and differentiation factor-8 (GDF8, also known as myostatin), isa secreted ligand belonging to the transforming growth factor-β (TGF-β)superfamily of growth factors. GDF8 plays a central role in thedevelopment and maintenance of skeletal muscle, acting as a negativeregulator of muscle mass. While the myostatin null mouse phenotypedemonstrates the importance of GDF8 in the control of muscle size duringdevelopment, muscle hypertrophy can also be elicited in adult musclethrough inhibition of GDF8 with neutralizing antibodies, decoyreceptors, or other antagonists. Administration of GDF8 neutralizingantibodies has been reported to result in muscle mass increases ofbetween 10 and 30%. The increased muscle mass seen is due to increasedfiber diameter as opposed to myofiber hyperplasia (fiber number). Anumber of studies have also reported increases in muscle strength orperformance commensurate with increased size including twitch andtetanic force. Use of a cleavage resistant version of the GDF8propeptide also leads to increased muscle size.

Other GDF8 antagonists have been used in adult mice with significanteffects on skeletal muscle mass. These include the extracellular portionof the Type II GDF8 receptor, ActRIIB, stabilized by fusion to an IgG Fcdomain (“ActRIIB-Fc”). The clinical molecule “ACE-031” is an example ofan ActRIIB-Fc molecule.

Although ActRIIB-Fc has been shown to increase muscle mass inexperimental animals, in human clinical trials this molecule was shownto cause various adverse side effects. For example, administration ofACE-031 to postmenopausal women in a Phase Ib ascending dose study wasshown to cause undesired increases in hemoglobin and decreases in FSHlevels. In addition, a Phase II study of ACE-031 in pediatric patientswith muscular dystrophy was discontinued due to adverse effectsincluding nose and gum bleeding. Dilated blood vessels are also observedin patients treated with ActRIIB-Fc.

Experiments have shown that the muscle growth-inducing effects ofActRIIB-Fc are attenuated but not eliminated in myostatin null mice,suggesting that ActRIIB-Fc exerts its muscle mass-inducing effects byantagonizing other ActRIIB ligand(s) in addition to GDF8. Other ligandsthat bind ActRIIB include Activin A, Activin B, Activin AB, Inhibin A,Inhibin B, GDF3, GDF11, Nodal, BMP2, BMP4, and BMP7.

BRIEF SUMMARY OF THE INVENTION

The present inventors hypothesized that the enhanced muscle growtheffects of ActRIIB-Fc, as well as its unwanted side effects, are due tothe binding of this molecule to additional ligands beside GDF8. Thus,the inventors sought to determine if it was possible to specificallyantagonize only certain ActRIIB ligands but not others in order toproduce the enhanced muscle growth effects of ActRIIB-Fc while at thesame time avoiding the unwanted adverse side effects associated withthis molecule. Through the experimentation set out in the Examplesherein, it was surprisingly discovered that significant muscle growthenhancement could be achieved by specifically antagonizing Activin A.Importantly, it was also determined that the desired therapeutic effectsof ActRIIB-Fc (e.g., enhanced skeletal muscle growth) could be achievedwithout unwanted side effects by specifically antagonizing GDF8 andActivin A but not antagonizing other ActRIIB ligands (e.g., GDF11, BMP9,BMP10, etc.).

Thus, according to one aspect of the present invention, a composition isprovided comprising a GDF8-specific binding protein and an ActivinA-specific binding protein. In certain embodiments, the GDF8-specificbinding protein is an anti-GDF8 antibody and/or the Activin A-specificbinding protein is an anti-Activin A antibody. According to a relatedaspect of the invention, an antigen-binding molecule is providedcomprising a GDF8-specific binding domain and an Activin A-specificbinding domain. In one embodiment of this aspect of the invention, theantigen-binding molecule is a bispecific antibody comprising a firstvariable domain that specifically binds GDF8 and a second variabledomain that specifically binds Activin A.

The present invention provides methods for increasing muscle mass orstrength in a subject by administering to the subject an ActivinA-specific binding protein. The present invention also provides methodsfor increasing muscle mass or strength in a subject by administering tothe subject a GDF8-specific binding protein and an Activin A-specificbinding protein, or by administering to the subject an antigen-bindingmolecule comprising a GDF8-specific binding domain and an ActivinA-specific binding domain. The methods according to this aspect of theinvention are useful for treating diseases or disorders associated withdecreased muscle mass, strength or power, including, e.g., cachexia,sarcopenia and other muscle-wasting conditions.

Other embodiments of the present invention will become apparent from areview of the ensuing detailed description.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Antigen-Specific Binding Proteins

The present invention relates to compositions comprisingantigen-specific binding proteins. More specifically, the presentinvention provides a composition comprising a GDF8-specific bindingprotein and an Activin A-specific binding protein.

As used herein, the expression “antigen-specific binding protein” meansa protein comprising at least one domain which specifically binds aparticular antigen. Exemplary categories of antigen-specific bindingproteins include antibodies, antigen-binding portions of antibodies,peptides that specifically interact with a particular antigen (e.g.,peptibodies), receptor molecules that specifically interact with aparticular antigen, and proteins comprising a ligand-binding portion ofa receptor that specifically binds a particular antigen.

The present invention includes antigen-specific binding proteins thatspecifically bind GDF8, “GDF8-specific binding proteins”. The term“GDF8” (also referred to as “growth and differentiation factor-8” and“myostatin”) means the protein having the amino acid sequence of SEQ IDNO:25 (mature protein). According to the present invention,GDF8-specific binding proteins specifically bind GDF8 but do not bindother ActRIIB ligands such as GDF3, BMP2, BMP4, BMP7, BMP9, BMP10,GDF11, Activin A, Activin B, Activin AB, Nodal, etc.

The present invention also includes antigen-specific binding proteinsthat specifically bind Activin A, i.e., “Activin A-specific bindingproteins”. Activins are homo- and heter-dimeric molecules comprising βAand/or βB subunits. The βA subunit has the amino acid sequence of SEQ IDNO:26 and the βB subunit has the amino acid sequence of SEQ ID NO:28.Activin A is a homodimer of two βA subunits; Activin B is a homodimer oftwo βB subunits; and Activin AB is a heterodimer of one βA subunit andone βB subunit. An Activin A-specific binding protein may be anantigen-specific binding protein that specifically binds the βA subunit.Since the βA subunit is found in both Activin A and Activin ABmolecules, an “Activin A-specific binding protein” can be anantigen-specific binding protein that specifically binds Activin A aswell as Activin AB (by virtue of its interaction with the βA subunit).Therefore, according to the present invention, an Activin A-specificbinding protein specifically binds Activin A, or Activin A and ActivinAB, but does not bind other ActRIIB ligands such as Activin B, GDF3,GDF8, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, Nodal, etc.

In the context of the present invention, molecules such as ActRIIB-Fc(e.g., “ACE-031”), which comprise the ligand-binding portion of theActRIIB receptor, are not considered “GDF8-specific binding proteins” or“Activin A-specific binding proteins” because such molecules bindmultiple ligands besides GDF8, Activin A and Activin AB.

Antigen-Binding Molecules with Two Different Antigen-Specific BindingDomains

The present invention also includes antigen-binding molecules comprisingtwo different antigen-specific binding domains. In particular, thepresent invention includes antigen-binding molecules comprising aGDF8-specific binding domain and an Activin A-specific binding domain.The term “antigen-specific binding domain,” as used herein, includespolypeptides comprising or consisting of: (i) an antigen-bindingfragment of an antibody molecule, (ii) a peptide that specificallyinteracts with a particular antigen (e.g., a peptibody), and/or (iii) aligand-binding portion of a receptor that specifically binds aparticular antigen. For example, the present invention includesbispecific antibodies with one arm comprising a first heavy chainvariable region/light chain variable region (HCVR/LCVR) pair thatspecifically binds GDF8 and another arm comprising a second HCVR/LCVRpair that specifically binds Activin A.

Specific Binding

The term “specifically binds” or the like, as used herein, means that anantigen-specific binding protein, or an antigen-specific binding domain,forms a complex with a particular antigen characterized by adissociation constant (K_(D)) of 500 pM or less, and does not bind otherunrelated antigens under ordinary test conditions. “Unrelated antigens”are proteins, peptides or polypeptides that have less than 95% aminoacid identity to one another. Methods for determining whether twomolecules specifically bind one another are well known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,and the like. For example, an antigen-specific binding protein or anantigen-specific binding domain, as used in the context of the presentinvention, includes molecules that bind a particular antigen (e.g.,GDF8, or Activin A and/or AB) or a portion thereof with a K_(D) of lessthan about 500 pM, less than about 400 pM, less than about 300 pM, lessthan about 200 pM, less than about 100 pM, less than about 90 pM, lessthan about 80 pM, less than about 70 pM, less than about 60 pM, lessthan about 50 pM, less than about 40 pM, less than about 30 pM, lessthan about 20 pM, less than about 10 pM, less than about 5 pM, less thanabout 4 pM, less than about 2 pM, less than about 1 pM, less than about0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less thanabout 0.05 pM, as measured in a surface plasmon resonance assay.

As used herein, an antigen-specific binding protein or antigen-specificbinding domain “does not bind” to a specified molecule (e.g., “does notbind GDF11”, “does not bind BMP9”, “does not bind BMP10”, etc.) if theprotein or binding domain, when tested for binding to the molecule at25° C. in a surface plasmon resonance assay, exhibits a K_(D) of greaterthan 1000 pM, or fails to exhibit any binding in such an assay orequivalent thereof.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore™ system(Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.).

The term “K_(D)”, as used herein, means the equilibrium dissociationconstant of a particular protein-protein interaction (e.g.,antibody-antigen interaction). Unless indicated otherwise, the K_(D)values disclosed herein refer to K_(D) values determined by surfaceplasmon resonance assay at 25° C.

Antibodies and Antigen-Binding Fragments of Antibodies

As indicated above, an antigen-specific binding protein can comprise orconsist of an antibody or antigen-binding fragment of an antibody.Furthermore, in the case of antigen-binding molecules comprising twodifferent antigen-specific binding domains, one or both of theantigen-specific binding domains may comprise or consist of anantigen-binding fragment of an antibody.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(L)) and alight chain constant region. The light chain constant region comprisesone domain (C_(L)1). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In different embodiments of the invention, the FRs of theantibodies of the invention (or antigen-binding portion thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H1)-C_(H)2; (V)V_(H)-C_(H1)-C_(H2)-C_(H)3; V_(H)-C_(H2)-C_(H)3; (Vii) V_(H)-C_(L);(Viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (X) V_(L)-C_(H)3; (xi)V_(L)-C_(H1)-C_(H)2; (xii) V_(L)-C_(H1)-C_(H2)-C_(H)3; (xiii)V_(L)-C_(H2)-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

The molecules of the present invention may comprise or consist of humanantibodies and/or recombinant human antibodies, or fragments thereof.The term “human antibody”, as used herein, includes antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. Human antibodies may nonetheless include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The molecules of the present invention may comprise or consist ofrecombinant human antibodies or antigen-binding fragments thereof. Theterm “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences.

Such recombinant human antibodies have variable and constant regionsderived from human germline immunoglobulin sequences. In certainembodiments, however, such recombinant human antibodies are subjected toin vitro mutagenesis (or, when an animal transgenic for human Igsequences is used, in vivo somatic mutagenesis) and thus the amino acidsequences of the V_(H) and V_(L) regions of the recombinant antibodiesare sequences that, while derived from and related to human germlineV_(H) and V_(L) sequences, may not naturally exist within the humanantibody germline repertoire in vivo.

Anti-GDF8 Antibodies and Antigen-Binding Fragments Thereof

In certain specific embodiments of the present invention, theGDF8-specific binding protein, or the GDF8-specific binding domain,comprises or consists of an anti-GDF8 antibody or antigen-bindingfragment thereof. Anti-GDF8 antibodies are mentioned in, e.g., U.S. Pat.Nos. 6,096,506; 7,320,789; 7,261,893; 7,807,159; 7,888,486; 7,635,760;7,632,499; in US Patent Appl. Publ. Nos. 2007/0178095; 2010/0166764;2009/0148436; and International Patent Appl. Publ. No. WO 2010/070094.Anti-GDF8 antibodies are also described in U.S. patent application Ser.No. 13/115,170, filed on May 25, 2011, and published as US 2011/______,including the antibodies designated 8D12, H4H1657N2, and H4H1669P. Anyof the anti-GDF8 antibodies mentioned and/or described in any of theforegoing patents or publications, or antigen-binding fragments thereof,can be used in the context of the present invention, so long as suchantibodies and/or antigen-binding fragments “specifically bind” GDF8, asthat expression is defined herein.

Table 1 sets forth the sequence identifiers of the HCVRs, LCVRs, andCDRs of certain non-limiting, exemplary anti-GDF8 antibodies that can beused in the context of the present invention.

TABLE 1 Antibody HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 8D12 1 23 4 5 6 7 8 H4H1657N2 9 10 11 12 13 14 15 16 H4H1669P 17 18 19 20 21 2223 24

Anti-Activin A Antibodies and Antigen-Binding Fragments Thereof

In certain specific embodiments of the present invention, the ActivinA-specific binding protein, or the Activin A-specific binding domain,comprises or consists of an antibody or antigen-binding fragment thereofthat specifically binds Activin A. In certain embodiments, the ActivinA-specific binding protein specifically binds the βA subunit. Anantigen-specific binding protein that specifically binds the βA subunitmay recognize both Activin A (βA/βA homodimer) and Activin AB (βA/βBheterodimer). Thus, according to the present invention, an ActivinA-specific binding protein may bind both Activin A and Activin AB (butnot Activin B). Anti-Activin A antibodies are mentioned in, e.g., USPatent Appl. Publ. No 2009/0234106. A particular anti-Activin A antibodyis designated “MAB3381,” and is available commercially from R&D Systems,Inc, Minneapolis, Minn. MAB3381 specifically binds Activin A (homodimer)as well as Activin AB (heterodimer). Any of the aforementionedanti-Activin A antibodies, or antigen-binding fragments thereof, can beused in the context of the present invention, so long as such antibodiesand/or antigen-binding fragments “specifically bind” Activin A and/orActivin AB, as defined herein.

Pharmaceutical Compositions and Methods of Administration

The present invention includes pharmaceutical compositions comprising aGDF8-specific binding protein and an Activin A-specific binding protein.The present invention also includes pharmaceutical compositionscomprising an antigen-binding molecule comprising a GDF8-specificbinding domain and an Activin A-specific binding domain. Thepharmaceutical compositions of the invention are formulated withsuitable carriers, excipients, and other agents that provide suitabletransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in, e.g., Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa. Suitable formulations include, forexample, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid(cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.Additional suitable formulations are also described in Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

Various delivery systems are known and can be used to administer thepharmaceutical compositions of the present invention, e.g.,encapsulation in liposomes, microparticles, microcapsules, recombinantcells capable of expressing the mutant viruses, receptor mediatedendocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).Methods of administration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical compositions of the presentinvention can be delivered in a controlled release system. In oneembodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRCCrit. Ref. Biomed. Eng. 14:201). In another embodiment, polymericmaterials can be used; see, Medical Applications of Controlled Release,Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe composition's target, thus requiring only a fraction of the systemicdose (see, e.g., Goodson, 1984, in Medical Applications of ControlledRelease, supra, vol. 2, pp. 115-138). Other controlled release systemsare discussed in the review by Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by knownmethods. For example, the injectable preparations may be prepared, e.g.,by dissolving, suspending or emulsifying the antibody or its saltdescribed above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc.

Dosage

The amount of active ingredient (e.g., anti-GDF8 antibodies andanti-Activin A antibodies) that can be administered to a subject is,generally, a therapeutically effective amount. As used herein, thephrase “therapeutically effective amount” means a dose ofantigen-specific binding proteins and/or antigen-binding molecules thatresults in a detectable increase in one or more of the followingparameters: body weight, muscle mass (e.g., tibialis anterior [TA]muscle mass, gastrocnemius [GA] muscle mass, quadriceps [Quad] musclemass, etc.), muscle strength/power, and/or muscle function. For example,a “therapeutically effective amount” of a GDF8-specific binding proteinand/or an Activin A-specific binding protein includes, e.g., an amountof GDF8-specific binding protein and/or Activin A-specific bindingprotein that, when administered to a test subject, causes an increase inTA or GA muscle mass of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%,50%, 60% or more, compared to control treated subjects, e.g., asillustrated in Example 1, herein.

In the case of antibodies of the present invention (e.g., anti-GDF8antibodies, anti-Activin A antibodies, or bispecific antibodies thatspecifically bind GDF8 and Activin A), a therapeutically effectiveamount can be from about 0.05 mg to about 600 mg; e.g., about 0.05 mg,about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg,about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570mg, about 580 mg, about 590 mg, or about 600 mg, of the respectiveantibody.

The amount of antibody of the present invention (e.g., anti-GDF8antibodies, anti-Activin A antibodies, or bispecific antibodies thatspecifically bind GDF8 and Activin A) contained within the individualdoses may be expressed in terms of milligrams of antibody per kilogramof patient body weight (i.e., mg/kg). For example, the anti-GDF8,anti-Activin A and/or anti-GDF8/anti-Activin A bispecific antibodies ofthe present invention may be administered to a patient at a dose ofabout 0.0001 to about 50 mg/kg of patient body weight (e.g. 0.5 mg/kg,1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg,7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, 10.0 mg/kg, 10.5mg/kg, 11.0 mg/kg, 11.5 mg/kg, etc.).

The compositions of the present invention may comprise equal amounts ofGDF8-specific binding protein and Activin A-specific binding protein.Alternatively, the amount of GDF8-specific binding protein in thecomposition may be less than or greater than the amount of ActivinA-specific binding protein. A person of ordinary skill in the art, usingroutine experimentation, will be able to determine the appropriateamounts of the individual components in the compositions of the presentinvention necessary to produce a desired therapeutic effect.

Therapeutic Methods

The present invention includes methods of treating conditions orafflictions which can be cured, alleviated or improved by increasingmuscle strength/power and/or muscle mass and/or muscle function in anindividual, or by favorably altering metabolism (carbohydrate, lipid andprotein processing) by specifically binding GDF8, and/or Activin A,and/or Activin AB, and not binding other ActRIIB ligands. For example,the present invention includes methods for increasing musclestrength/power and/or muscle mass and/or muscle function in a subject,or for treating a disease or disorder characterized by decreased musclemass or strength in a subject, by administering to the subject anActivin A-specific binding protein. The present invention also includesmethods for increasing muscle strength/power and/or muscle mass and/ormuscle function in a subject, or for treating a disease or disordercharacterized by decreased muscle mass or strength in a subject, byadministering to the subject a GDF8-specific binding protein and anActivin A-specific binding protein. Any of the GDF8-specific bindingproteins and/or Activin A-specific binding proteins disclosed orreferred to herein can be used in the context of these aspects of theinvention. For example, the therapeutic methods of the present inventioninclude administering to a subject an anti-GDF8 antibody and/or ananti-Activin A antibody.

In methods which comprise administering a GDF8-specific binding proteinand an Activin A-specific binding protein to a subject, theGDF8-specific binding protein and the Activin A-specific binding proteinmay be administered to the subject at the same or substantially the sametime, e.g., in a single therapeutic dosage, or in two separate dosageswhich are administered simultaneously or within less than about 5minutes of one another. Alternatively, the GDF8-specific binding proteinand the Activin A-specific binding protein may be administered to thesubject sequentially, e.g., in separate therapeutic dosages separated intime from one another by more than about 5 minutes.

The present invention also includes methods for increasing musclestrength/power and/or muscle mass and/or muscle function in a subject,or for treating a disease or disorder characterized by decreased musclemass or strength in a subject, by administering to the subject anantigen-binding molecule comprising a GDF8-specific binding domain andan Activin A-specific binding domain. Any of the antigen-bindingmolecules disclosed or referred to herein can be used in the context ofthis aspect of the invention. For example, the therapeutic methods ofthe present invention include administering to a subject a bispecificantibody comprising a first variable domain comprising a HCVR/LCVR pairthat specifically binds GDF8 and a second variable domain comprising aHCVR/LCVR pair that specifically binds Activin A.

The compositions of the present invention may be administered to asubject along with one or more additional therapeutic agents, including,e.g., growth factor inhibitors, immunosuppressants, anti-inflammatoryagents, metabolic inhibitors, enzyme inhibitors, andcytotoxic/cytostatic agents. The additional therapeutic agent(s) may beadministered prior to, concurrent with, or after the administration ofthe GDF8- and Activin A-specific binding proteins of the presentinvention.

Exemplary diseases, disorders and conditions that can be treated withthe compositions of the present invention include, but are not limitedto, sarcopenia, cachexia (either idiopathic or secondary to otherconditions, e.g., cancer, chronic renal failure, or chronic obstructivepulmonary disease), muscle injury, muscle wasting and muscle atrophy,e.g., muscle atrophy or wasting caused by or associated with disuse,immobilization, bed rest, injury, medical treatment or surgicalintervention (e.g., hip fracture, hip replacement, knee replacement,etc.) or by necessity of mechanical ventilation. The compositions of theinvention may also be used to treat, prevent or ameliorate diseases suchas cancer, obesity, diabetes, arthritis, multiple sclerosis, musculardystrophy, amyotrophic lateral sclerosis, Parkinson's disease,osteoporosis, osteoarthritis, osteopenia, metabolic syndromes(including, but not limited to diabetes, obesity, nutritional disorders,organ atrophy, chronic obstructive pulmonary disease, and anorexia).

Avoidance of Side Effects

The present invention includes methods for increasing musclestrength/power and/or muscle mass and/or muscle function in a subject,or for treating a disease or disorder characterized by decreased musclemass or strength in a subject, without causing adverse side effectsassociated with the administration of molecules which bind multiple(e.g., 3 or more) ActRIIB ligands. For example, the clinical moleculereferred to as ACE-031 (Acceleron Pharma, Inc., Cambridge, Mass.) is amultimer consisting of the extracellular portion of ActRIIB fused to anIgG Fc domain (this molecule is also referred to herein as“ActRIIB-Fc”). ActRIIB-Fc binds GDF8 as well as other ActRIIB ligandssuch as, e.g., Activin A, Activin B, GDF11, BMP9, BMP10, and TGFβ, andis known to cause various adverse side effects when administered tohuman patients. Significantly, the present inventors have unexpectedlydiscovered that specifically inhibiting GDF8 and Activin A (e.g., byadministering an anti-GDF8 antibody and an anti-Activin A antibody),while not inhibiting other ActRIIB ligands such as Activin B, GDF11,BMP9, BMP10, and TGFβ, results in an increase in muscle mass that is atleast equivalent to that observed by administration of ActRIIB-Fc,without causing the adverse side effects associated with non-specificActivin binding agents such as ActRIIB-Fc.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of the compositions of the present invention (e.g., compositionscomprising GDF8- and/or Activin A-specific binding proteins orantigen-binding molecules comprising a GDF8-specific binding domain andan Activin A-specific binding domain), may be administered to a subjectover a defined time course. The methods according to this aspect of theinvention comprise sequentially administering to a subject multipledoses of the compositions of the present invention. As used herein,“sequentially administering” means that each dose of the compositions ofthe present invention are administered to the subject at a differentpoint in time, e.g., on different days separated by a predeterminedinterval (e.g., hours, days, weeks or months). The present inventionincludes methods which comprise sequentially administering to thepatient an initial dose of a composition of the present invention,followed by one or more secondary doses of the composition, andoptionally followed by one or more tertiary doses of the composition.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the compositions of thepresent invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount of activeingredient(s), but will generally differ from one another in terms offrequency of administration. In certain embodiments, however, the amountof active ingredient(s) contained in the initial, secondary and/ortertiary doses will vary from one another (e.g., adjusted up or down asappropriate) during the course of treatment.

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, or more) days after the immediately preceding dose.The phrase “the immediately preceding dose,” as used herein, means, in asequence of multiple administrations, the dose(s) of the compositions ofthe present invention which are administered to a subject prior to theadministration of the very next dose in the sequence with no interveningdoses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof the compositions of the present invention. For example, in certainembodiments, only a single secondary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) secondary doses are administered to the patient. Likewise, incertain embodiments, only a single tertiary dose is administered to thepatient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8,or more) tertiary doses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to29 days after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 1 to 60days after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Specific Inhibition of GDF8 and Activin A Causes SynergisticIncreases in Skeletal Muscle Mass Introduction

ActRIIB-Fc is a GDF8 antagonist consisting of the extracellular portionof the ActRIIB receptor, stabilized by fusion to an IgG Fc domain.ActRIIB-Fc has been shown to increase muscle mass in mice to a greaterextent than anti-GDF8 antibodies. The present inventors hypothesizedthat the enhanced activity of ActRIIB-Fc could potentially be due to itsability to bind additional ActRIIB ligands besides GDF8. In particular,it was proposed that antagonism of Activin A, in addition to antagonismof GDF8, might cause greater increases in skeletal muscle mass than whathas been observed in animals treated with anti-GDF8 antibodies alone.Thus, the present Example was designed to determine whether specificinhibition of GDF8 and Activin A can increase skeletal muscle mass to anextent that is at least equivalent to the increase observed usingActRIIB-Fc.

Results and Discussion

The extent of skeletal muscle hypertrophy induced by administration ofActRIIB-Fc was compared to the effect of administration of aGDF8-specific antibody, an Activin A specific antibody, or a combinationof an anti-GDF8+anti-Activin A antibody. The ActRIIB-Fc construct usedin this Example has the amino acid sequence of SEQ ID NO:27. Theanti-GDF8 antibody used in this Example is the antibody designatedH4H1657N2 (see Table 1). The anti-Activin A antibody used in thisExample is the antibody designated MAB3381 (available from R&D Systems,Inc., Minneapolis, Minn.). An isotype-matched (hIgG4) antibody was usedas negative control.

Briefly, 25 male CB17 SCID mice at approximately 10 weeks of age, weredivided evenly according to body weight into 5 groups based on treatment(Isotype Control mAb, ActRIIB-Fc, H4H1657N2, MAB3381, orH4H1657N2+MAB3381). Reagents were administered subcutaneously at a doseof 10 mg/kg twice for the first week (on day 0 and day 3) and once aweek for the following three weeks (on day 7, day 14 and day 21). On day28, mice were euthanized and weighed, and the tibialis anterior (TA)muscles, and the gastrocnemius (GA) muscles, were dissected and weighed.Tissues were normalized to starting body weight, and percent change inweight over the isotype-matched (hIgG4) control antibody was calculated.Results are summarized in Table 2 and are expressed as percent increaseover negative control±standard error of the mean.

TABLE 2A Isotype H4H1657N2 MAB3381 H4H1657N2 + Control ActRIIB-Fc(anti-GDF8) (Anti-Activin A) MAB3381 Dose 10 mg/kg 10 mg/kg 10 mg/kg 10mg/kg 10 mg/kg (each) Body 0.00 ± 1.18 14.83 ± 4.36  7.88 ± 2.10  4.52 ±1.02 16.08 ± 1.91 Weight TA Muscle 0.00 ± 2.90 44.88 ± 5.35 22.42 ± 1.6519.09 ± 2.04 55.13 ± 5.16 GA Muscle 0.00 ± 2.13 34.25 ± 6.97 24.17 ±1.84 14.02 ± 0.91 41.72 ± 3.63

In order to confirm that muscle hypertrophy was the result of anincrease in muscle fiber size, the tibialis anterior (TA) muscle wasembedded in OCT and isopentane-frozen for histological examination andimmunohistochemical labeling. Cross-sections of the TA muscle werestained with anti-laminin antibody to outline the muscle fiber, and theaverage cross-sectional-area (CSA) was determined by using an imaginganalysis system. Results of two independent experiments (Exp#1 andExp#2) are summarized in Table 2B. All data are expressed as means±thestandard error of the mean.

TABLE 2B Isotype H4H1657N2 MAB3381 H4H1657N2 + Control ActRIIB-Fc(anti-GDF8) (Anti-Activin A) MAB3381 CSA (μm²) 1800.1 ± 78.3 2488.7 ±116.6 1987.2 ± 72.1  1962.6 ± 157.1 2435.4 ± 119.7 Exp#1 CSA (μm²)1702.7 ± 50.9 2571.9 ± 123.3 2006.3 ± 133.9 1690.9 ± 78.9  2452.6 ±110.3 Exp#2

As shown in Table 2A, ActRIIB-Fc induced significant hypertrophy in allmuscles examined, with increases of 44.88±5.35% in TA muscle mass, and34.25±6.97% in GA muscle mass. Treatment with H4H1657N2 (anti-GDF8), orMAB3381 (anti-Activin A) alone also induced significant hypertrophy inTA muscle mass (22.42±1.65% and 19.09±2.04%, respectively) and GA musclemass (24.17±1.84 and 14.02±0.91%, respectively) but not as pronounced asActRIIB-Fc. However, the combination of H4H1657N2 and MAB3381 inducedincreases in TA (55.13±5.16%) and GA (41.72±3.63%), that were evengreater than what was observed in ActRIIB-hFc-treated animals.Furthermore, it was confirmed that the muscle hypertrophy observed wasthe result of an increase in muscle fiber size (see Table 2B).

Importantly, the extent of increases in body weight, TA muscle, and GAmuscle for the anti-GDF8+anti-Activin A combination were substantiallygreater than the sums of the increases in these parameters observed inthe anti-GDF8 plus anti-Activin A monotherapy subjects. Thus, combinedinhibition of GDF8 and Activin A produces synergistic increases in bodyweight and skeletal muscle mass, and these increases are more pronouncedthan what is observed in ActRIIB-Fc-treated animals. Moreover, asdemonstrated in the following Example, the increases in body weight andskeletal muscle mass in animals that are treated with GDF8- and ActivinA-specific binding agents, can be achieved without causing the adverseside effects observed with molecules such as ActRIIB-Fc.

Example 2 Specific Antagonism of GDF8 and Activin A does not CauseAdverse Side

Effects Associated with Non-Specific Activin Ligand Binding Agents

Background

ActRIIB-Fc binds multiple ActRIIB ligands and causes significant sideeffects. The present Example demonstrates that the adverse side effectsassociated with ActRIIB-Fc can be avoided by selectively antagonizingonly certain ActRIIB ligands, namely GDF8 and/or Activin A. Inparticular, biomarker, protein expression studies, and in vivo red bloodcell characteristics (i.e., elevated endoglin levels and increased redcell distribution width), which appear to be linked to ActRIIB-Fc sideeffects in humans, were only seen in animals treated with ActRIIB-Fc,but not in animals treated with anti-GDF8 antibody, anti-Activin Aantibody or a combination of anti-GDF8 and anti-Activin A antibodies.Thus, taken together, the results below show that specific antagonism ofGDF8 and/or Activin A, without antagonizing other ActRIIB ligands suchas Activin B, GDF11, BMP9, BMP10, and/or TGFβ, does not cause theundesired phenotypes associated with ActRIIB-Fc.

Results and Discussion

Hematology studies were conducted using mice treated with ActRIIB-Fc(SEQ ID NO:27), H4H1657N2 (anti-GDF8), MAB3381 (anti-Activin A), or acombination of H4H1657N2+MAB3381 according to the dosing scheduledescribed in Example 1 (i.e., 10 mg/kg twice for the first week [on day0 and day 3] and once a week for the following three weeks [on day 7,day 14 and day 21]). Specifically, hemoglobin levels and red blood celldistribution width (RDW) (an indicator of certain blood disorders suchas anemia) were measured from blood samples taken from mice treated withthe various agents. RDW results (normalized to Isotype Control values)are summarized in Table 3.

TABLE 3 Isotype H4H1657N2 MAB3381 H4H1657N2 + Control ActRIIB-Fc(anti-GDF8) (Anti-Activin A) MAB3381 Dose 10 mg/kg 10 mg/kg 10 mg/kg 10mg/kg 10 mg/kg (each) % RDW 0.0 ± 1.8 19.1 ± 2.1 −1.6 ± 0.7 4.4 ± 0.5−1.9 ± 1.2

After 28 days of treatment, none of the groups had a significantincrease in Hb levels. However, as shown in Table 3, mice treated withActRIIB-Fc showed a significant increase in red blood cell distributionwidth (RDW), which reflects the extent of size variation of red bloodcells in a sample. Surprisingly, mice treated with anti-GDF8 antibody,anti-Activin A antibody, or the combination of anti-GDF8+anti-Activin Aantibodies, did not exhibit an appreciable degree of increase in % RDWas compared to isotype control-treated mice. These experiments thereforedemonstrate that antagonism of GDF8 or Activin A alone, or thecombination of anti-GDF8 antibody+anti-Activin A antibodies, do notcause the hematological phenotypes that are observed with ActRIIB-Fctreatment.

To further investigate the differences in side effects betweenActRIIB-Fc-treated subjects and anti-GDF8+anti-Activin Acombination-treated subjects, microarray and protein expression studieswere conducted.

Microarray analysis was conducted on skeletal muscle samples from micetreated with isotype control, ActRIIB-Fc, and H4H1657N2 (anti-GDF8).From these experiments, a set of genes was identified that is uniquelyaffected by ActRIIB-Fc. Of particular interest was the up-regulation ofEndoglin mRNA levels in skeletal muscle in samples fromActRIIB-Fc-treated subjects. Endoglin is a transmembrane proteinexpressed primarily in endothelial cells and interacts and promotessignaling through receptors of the TGFβ family (ALK1) in response toTGFβ, BMP9, or BMP10. Signaling mediated by Alk1 and Endoglin inendothelial cells is required for maintaining normal vascularstructures. Mutations in the Alk1 and Endoglin genes in humans causesHereditary Haemorrhagic Talangiectasia (HHT). Patients suffering HHTdisplay a vascular phenotype including dilated blood vessels, andbleeding in the nasal, oral, and gastrointentinal mucosa. Thus, elevatedEndoglin levels caused by ActRIIB-Fc potentially reflect at least someof the adverse side effects observed in patients treated with thistherapeutic agent.

Next, experiments were conducted to confirm that the changes observed inEndoglin mRNA levels were also reflected at the protein expression levelusing muscle samples from the previous experiment. Quantitative Westernblot analysis of Endoglin protein levels was conducted on samples frommice treated with isotype control, ActRIIB-Fc, H4H1657N2 (anti-GDF8),MAB3381 (anti-Activin A), and the H4H1657N2+MAB3381 combination.Expression of Endoglin was normalized by the endothelial cell markerCD31 to confirm that ActRIIB-hFc treatment does not increase theendothelial compartment. Results (normalized to Isotype Control values)are summarized in Table 4.

TABLE 4 Isotype H4H1657N2 MAB3381 H4H1657N2 + Control ActRIIB-Fc(anti-GDF8) (Anti-Activin A) MAB3381 Dose 10 mg/kg 10 mg/kg 10 mg/kg 10mg/kg 10 mg/kg (each) Endoglin O.D. 0.0 ± 1.4 88.4 ± 2.9  5.5 ± 2.1 4.7± 5.0 1.4 ± 7.6 CD31 O.D. 0.0 ± 4.2  2.2 ± 6.2 −6.8 ± 4.0 −2.1 ± 9.3  −19.3 ± 6.7  O.D. Ratio 0.0 ± 5.6 84.5 ± 8.3 13.1 ± 2.6 7.1 ± 5.1 25.5 ±1.0 

As shown in Table 4, levels of Endoglin protein were significantlyelevated in the ActRIIB-hFc group, but not in the anti-GDF8 oranti-Activin A-treated groups. Interestingly, Endoglin levels were alsonot elevated in the anti-GDF8+anti-Activin A combination-treated group.

SUMMARY AND CONCLUSIONS

The results presented in the prior Example (Example 1) show that thecombination of anti-GDF8+anti-Activin A treatment can produce musclehypertrophy effects that are at least equivalent to those observed withActRIIB-Fc treatment. The present Example 2 shows that indicators of theadverse side effects of ActRIIB-Fc treatment, such as increased RDW andelevated Endoglin expression, are not observed with anti-GDF8,anti-Activin A, or anti-GDF8+anti-Activin A combination treatment. Thus,the present inventors have surprisingly discovered that treatment with aGDF8-specific binding protein, or an Activin A-specific binding protein,or a combination of a GDF8-specific binding protein and an ActivinA-specific binding protein provide highly efficacious methods forincreasing muscle mass and strength that avoid the adverse side effectscaused by ActRIIB-Fc.

Example 3 Effects of GDF8 and Activin A Antagonists on Wound Healing

Pharmaceutical agents which function to increase muscle mass andstrength, such as GDF8 antagonists and Activin A antagonists, haveutility in settings in which patients have undergone surgery (or willundergo surgery), e.g., for joint replacement or repair, etc. As such,agents that are administered to promote the rescue of muscle mass wouldideally not interfere with other aspects of surgical recovery such aswound healing.

Accordingly, experiments were conducted to determine the effects of GDF8blockade, Activin A blockade, and combinations thereof, on woundhealing, as compared to the effects of ActRIIB-Fc treatment. Thesestudies were carried out in SCID mice. In particular, the effects ofH4H1657N2 (anti-GDF8) and MAB3381 (anti-Activin A) administration onwound healing, as single treatments or in combination with one another,were compared to the wound healing effects of the more broadly-actingdecoy receptor ActRIIB-hFc (SEQ ID NO:27). Briefly, circular skinexcisional wounds were made on the left abdominal flank of 30 male SCIDmice approximately 7-8 weeks. The animals were divided into fivetreatment groups (n=6 per group) each receiving five subcutaneousinjections of an isotype control antibody, ActRIIB-hFc, H4H1657N2,MAB3381, or H4H1657N2+MAB3381. All reagents were administered at 10mg/kg every 3-4 days. The first dose was given the day before woundingthe animals, and the last one was given two days before terminating thestudy on day 14. Digital images of the wound were taken on day 0 (day ofwounding), 6, 9, 12, and 14, and the excision wound size change wasmeasured and compared to the isotype control group. Results aresummarized in Table 5. All data are expressed as mean total woundsize±the standard error of the mean.

TABLE 5 Treatment Days After Wounding (total wound size mm²) (10 mg/kgevery 3-4 days) Day 0 Day 6 Day 9 Day 12 Day 14 Isotype control 59.95 ±2.25 31.92 ± 3.43 18.75 ± 3.67  9.46 ± 2.25  7.66 ± 1.51 ActRIIB-hFc61.30 ± 1.78 49.07 ± 2.74 30.50 ± 2.74 17.60 ± 1.53 14.90 ± 1.25H4H1657N2 (anti-GDF8) 64.98 ± 2.56 30.69 ± 5.3  16.36 ± 3.27  7.71 ±1.93  6.98 ± 1.32 MAB3381 (anti-Activin A) 62.07 ± 2.94 35.58 ± 4.9623.38 ± 3.3  13.67 ± 2.19 11.01 1.45 H4H1657N2 + MAB3381 61.08 ± 2.5431.85 ± 2.83 17.68 ± 2.17 10.89 ± 1.74 9.09 ± 1.4

An analysis of wound size at the end of the experiment, as shown inTable 5, revealed that treatment with H4H1657N2, MAB3381, orH4H1657N2+MAB3381 resulted in no significant difference in wound size atany time after the initial excision as compared to the isotype controlgroup. By contrast, ActRIIB-hFc significantly delayed wound closure asindicated by the larger wound size at days 6, 9, 12, and 14 as comparedto the wound size in mice treated with H4H1657N2, MAB3381,H4H1657N2+MAB3381, or the isotype control.

This experiment demonstrates that therapeutic treatments involving GDF8antagonism, Activin A antagonism, or GDF8+Activin A dual antagonism, donot significantly impair wound healing, whereas the less specificantagonist ActRIIB-hFc does significantly impair wound healing.Accordingly, the present Example provides further support for the notionthat specific antagonism of GDF8 and Activin A can produce enhancedmuscle mass and function, similar to what is seen with ActRIIb-hFctreatment, but without the adverse side effects associated withActRIIb-hFc treatment.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. A composition comprising a GDF8-specific bindingprotein and an Activin A-specific binding protein.
 2. The composition ofclaim 1, wherein the GDF8-specific binding protein is an anti-GDF-8antibody or antigen-binding fragment thereof.
 3. The composition ofclaim 1, wherein the Activin A-specific binding protein is ananti-Activin A antibody or antigen-binding fragment thereof.
 4. Thecomposition of claim 1, wherein the GDF8-specific binding protein is ananti-GDF-8 antibody or antigen-binding fragment thereof, and wherein theActivin A-specific binding protein is an anti-Activin A antibody orantigen-binding fragment thereof.
 5. The composition of claim 2, whereinthe anti-GDF8 antibody or antigen-binding fragment thereof comprises theheavy chain complementarity determining regions (HCDRs) of a heavy chainvariable region (HCVR) comprising SEQ ID NO:9, and the light chaincomplementarity determining regions (LCDRs) of a light chain variableregion (LCVR) comprising SEQ ID NO:13.
 6. The composition of claim 5,wherein the anti-GDF8 antibody or antigen-binding fragment thereofcomprises three HCDRs comprising SEQ ID NO:10, SEQ ID NO:11, and SEQ IDNO:12, and three LCDRs comprising SEQ ID NO:14, SEQ ID NO:15, and SEQ IDNO:16.
 7. An antigen-binding molecule comprising a GDF8-specific bindingdomain and an Activin A-specific binding domain.
 8. The antigen-bindingmolecule of claim 7, wherein the GDF8-specific binding domain comprisesa heavy chain variable region (HCVR) and a light chain variable region(LCVR).
 9. The antigen-binding molecule of claim 7, wherein the ActivinA-specific binding domain comprises a heavy chain variable region (HCVR)and a light chain variable region (LCVR).
 10. The antigen-bindingmolecule of claim 8, wherein the HCVR comprises three heavy chaincomplementarity determining regions (HCDRs) comprising SEQ ID NO:10, SEQID NO:11, and SEQ ID NO:12, and wherein the LCVR comprises three lightchain complementarity determining regions (LCDRs) comprising SEQ IDNO:14, SEQ ID NO:15, and SEQ ID NO:16.
 11. The antigen-binding moleculeof claim 7, wherein the GDF8-specific binding domain comprises a heavychain variable region (HCVR) and a light chain variable region (LCVR),and wherein the Activin A-specific binding domain comprises a heavychain variable region (HCVR) and a light chain variable region (LCVR).12. The antigen-binding molecule of claim 11, wherein theantigen-binding molecule is a bispecific antibody.
 13. A method forincreasing muscle mass or strength in a subject, the method comprisingadministering to the subject an Activin A-specific binding protein. 14.The method of claim 13, wherein the Activin A-specific binding proteinis an anti-Activin A antibody or antigen-binding fragment thereof.
 15. Amethod for increasing muscle mass or strength in a subject, the methodcomprising administering to the subject a GDF8-specific binding proteinand an Activin A-specific binding protein.
 16. The method of claim 15,wherein the GDF8-specific binding protein is an anti-GDF8 antibody orantigen-binding fragment thereof.
 17. The method of claim 15, whereinthe Activin A-specific binding protein is an anti-Activin A antibody orantigen-binding fragment thereof.
 18. The method of claim 15, whereinthe GDF8-specific binding protein is an anti-GDF8 antibody orantigen-binding fragment thereof, and wherein the Activin-A-specificbinding protein is an anti-Activin A antibody or antigen-bindingfragment thereof.
 19. The method of claim 16, wherein the anti-GDF8antibody or antigen-binding fragment thereof comprises the heavy chaincomplementarity determining regions (HCDRs) of a heavy chain variableregion (HCVR) comprising SEQ ID NO:9, and the light chaincomplementarity determining regions (LCDRs) of a light chain variableregion (LCVR) comprising SEQ ID NO:13.
 20. The method of claim 19,wherein the anti-GDF8 antibody or antigen-binding fragment thereofcomprises three HCDRs comprising SEQ ID NO:10, SEQ ID NO:11, and SEQ IDNO:12, and three LCDRs comprising SEQ ID NO:14, SEQ ID NO:15, and SEQ IDNO:16.
 21. A method for increasing muscle mass or strength in a subject,the method comprising administering to the subject an antigen-bindingmolecule comprising a GDF8-specific binding domain and an ActivinA-specific binding domain.
 22. The method of claim 21, wherein theGDF8-specific binding domain comprises a heavy chain variable region(HCVR) and a light chain variable region (LCVR).
 23. The method of claim21, wherein the Activin A-specific binding domain comprises a heavychain variable region (HCVR) and a light chain variable region (LCVR).24. The method of claim 22, wherein the HCVR comprises three heavy chaincomplementarity determining regions (HCDRs) comprising SEQ ID NO:10, SEQID NO:11, and SEQ ID NO:12, and wherein the LCVR comprises three lightchain complementarity determining regions (LCDRs) comprising SEQ IDNO:14, SEQ ID NO:15, and SEQ ID NO:16.
 25. The method of claim 21,wherein the GDF8-specific binding domain comprises a heavy chainvariable region (HCVR) and a light chain variable region (LCVR), andwherein the Activin A-specific binding domain comprises a heavy chainvariable region (HCVR) and a light chain variable region (LCVR).
 26. Themethod of claim 25, wherein the antigen-binding molecule is a bispecificantibody.
 27. A method for treating a disease or disorder characterizedby decreased muscle mass or strength, the method comprisingadministering to a subject in need thereof an Activin A-specific bindingprotein.
 28. A method for treating a disease or disorder characterizedby decreased muscle mass or strength, the method comprisingadministering to a subject in need thereof a GDF8-specific bindingprotein and an Activin A-specific binding protein.
 29. A method fortreating a disease or disorder characterized by decreased muscle mass orstrength, the method comprising administering to a subject in needthereof an antigen-binding molecule comprising a GDF8-specific bindingdomain and an Activin A-specific binding domain.